Sirtuin 1 (SIRT1) is a protein deacetylase that has anti-diabetic effects and promotes healthy aging. Consequently, SIRT1 is a major therapeutic target to reduce metabolic diseases and increase lifespan. Similar to SIRT1, the Mediterranean Lifestyle, defined as high levels of physical activity in conjunction with the Mediterranean Diet, significantly reduces the risk of metabolic and aging-associated diseases. The discovery that resveratrol, a bioactive compound in red wine, activates SIRT1 was thought to explain a major biological mechanism of the Mediterranean Diet. However, this enthusiasm has been tempered by data showing that the effects of resveratrol on SIRT1 are indirect and non-specific. Another plausible, yet unexplored, mechanism involves oleate, the main monounsaturated fatty acid in olive oil. The mechanism by which oleate produces its well-established metabolic benefits is not well understood and whether specific dietary lipids may interact with physical activity to affect metabolism remains unknown. This knowledge gap presents an opportunity to further define the underlying biology driving the metabolic benefit of the Mediterranean Lifestyle/Diet. Based upon strong preliminary data linking lipolytically-derived oleate to SIRT1 activation, the objective of this proposal is to characterize the physiological significance of lipolysis and oleate metabolism in insulin resistance and energy metabolism and to identify the cellular factors that influence ATGL-mediated oleate signaling. We will test the hypothesis that ATGL-catalyzed lipolysis, or factors that influence lipolysis such as exercise, will synergize with a major dietary source of oleate, olive oil, to promote insulin sensitivity, oxidative metabolism and metabolic health via SIRT1 activation. To test our hypothesis, we will conduct the following specific aims: 1) to determine the synergistic effects of global ATGL overexpression, dietary olive oil and SIRT1 on energy metabolism and insulin resistance, 2) to determine the synergistic effects of exercise, dietary olive oil and SIRT1 on energy metabolism and insulin resistance, and 3) to determine how proteins involved in oleate metabolism influence ATGL-mediated SIRT1 activation. Aim 1 will characterize interactions between dietary oleate composition and global ATGL overexpression on insulin resistance and energy metabolism in db/db and diet-induced obese mice. Aim 2 will explore the synergy between exercise and dietary oleate as a means to promote metabolic health. Aim 3 will employ cell models to characterize changes in oleate metabolism that influence its regulation of SIRT1. These studies are innovative in that they will answer novel questions about SIRT1 regulation, exercise and diet and define a major mechanism through which the Mediterranean Lifestyle elicits its effects. This work is significant as it opens new directions for dietary, behavioral and pharmaceutical approaches to treat insulin resistance and its comorbidities.